This is an application for continuation of a 10 year old project whose goal is to understand the regulation and function of different signaling responses controlled by the I?B kinase (IKK) complex and its three subunits: IKKa, IKK, and IKK?. Progress during the previous project period has been considerable - in addition to improved understanding of IKK regulation and its role in NF-?B activation, we have elucidated the roles of the two IKK catalytic subunits (IKKa and IKK) in the control of innate immunity and inflammation as well as programmed cell death, cancer, chronic inflammatory disorders and autoimmunity. During the course of these studies, we made several unanticipated and rather surprising findings that we plan to pursue in greater detail during the next project period. These findings include the identification of the Hif1a gene, which encodes the hypoxia regulated HIF-1a subunit of the HIF-1 transcription factor, as an IKK and NF-?B regulated gene. These results suggest that in addition to its key role in suppression of apoptosis and activation of host defense and inflammation, NF-?B is also a critical regulator of the hypoxic response. In addition to studying the regulation of IKK? activity by hypoxia we will conduct studies that should clarify the pathophysiological importance of these findings in the control of innate immunity and tumor-elicited inflammation. Another unanticipated finding is the involvement of IKK?(NEMO), the IKK regulatory subunit, in activation of the JNK and p38 MAPK pathways in B cells stimulated via CD40 or BAFF receptor (BAFF-R). We will study the mechanism by which IKK? controls the activation of these pathways through formation of a signaling complex that includes TRAF2 and MEKK1, which are E3 ubiquitin ligases, and UBC13, an E2 ubiquitin conjugating enzyme. In addition we will continue to study the role of TRAF2 and its relative TRAF3 in controlling the activation of the IKK-dependent alternative NF-?B signaling pathway in response to engagement of CD40, RANK, BAFF-R and lymphotoxin(LT) a: receptor (LTR). We will focus on the role of TRAF2 and TRAF3 in controlling the turnover of NIK, a protein kinase responsible for IKKa activation. A third unexpected finding that will be followed up on is a role for activated nuclear IKKa in transcriptional regulation that is not related to its already known functions in regulation of the classical and alternative NF-?B signaling pathways. Although we first found this novel function of nuclear IKK in prostate cancer we will study its relevance to signaling by IKKa-activating receptors in cell types present within secondary lymphoid organs, such as the spleen, whose development and function are IKKa-dependent. As before, these studies will advance both our basic understanding of IKK signaling as well as its varied and wide-reaching pathophysiological functions. Project Narrative: This is an application for continuation of a 10 year old project focused on the regulation and function of the I?B kinase (IKK) complex, a master regulator of immunity, host defense and cell survival. The new studies will follow unexpected findings made during the previous project period that pertain to roles of IKK subunits in control of the hypoxic response during microbial infections and tumor-elicited inflammation and regulation of the alternative NF-?B signaling pathway, which is important for lymphoid organ development and function, as well as the pathogenesis of autoimmunity. Other new findings that will be studied in further detail are the role of the IKK? subunit in signaling to MAPK cascades during activation of antibody-producing B cells and novel nuclear functions for IKKa in cells of secondary lymphoid organs, which are important players in immunity and inflammatory diseases.